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| United States Patent |
5,096,924
|
|
Ishizuka
, et al.
|
March 17, 1992
|
Anticancer antibiotic MI43-37F11 substance
Abstract
As a new antibiotic is provided a compound, now nominated as MI43-37F11
substance, which has formula
##STR1##
This MI43-37F11 substance has an antitumor activity, an activity to
enhance the production of interleukin-1 in vivo in a mammalian, and an
activity to activate a macrophage in vivo in a mammalian. MI43-37F11
substance may be produced by cultivation of Streptoverticillium
eurocidicum MI43-37F11 strain identified as FERM BP-2783.
| Inventors:
|
Ishizuka; Masaaki (Mishima, JP);
Kumagai; Hiroyuki (Numazu, JP);
Sawa; Tsutomu (Ayase, JP);
Naganawa; Hiroshi (Tokyo, JP);
Iinuma; Hironobu (Wako, JP);
Isshiki; Kunio (Fujisawa, JP);
Hamada; Masa (Tokyo, JP);
Maeda; Kenji (Tokyo, JP);
Takeuchi; Tomio (Tokyo, JP)
|
| Assignee:
|
501 Azidan Hojin Biseibutsu Kagaku Kenkyu Kai (Tokyo, JP)
|
| Appl. No.:
|
531109 |
| Filed:
|
May 31, 1990 |
Foreign Application Priority Data
| Current U.S. Class: |
514/456; 435/123; 549/289 |
| Intern'l Class: |
A61K 031/35; C07D 311/76 |
| Field of Search: |
549/289
514/456
|
References Cited
Other References
Furutani et al., CA 87:113776x.
Umezawa et al., CA 89:197337g.
|
Primary Examiner: Fan; Jane T.
Attorney, Agent or Firm: Larson and Taylor
Claims
We claim:
1. An antibiotic, MI43-37F11 substance which is a compound having the
formula
##STR7##
2. A pharmaceutical composition for inhibiting a carcinoma sensitive to the
MI4337F11 substance comprising the MI43-37F11 substance having the formula
as defined in claim 1, as active ingredient, in an amount effective to
inhibit said carcinoma in association with a pharmaceutically acceptable
carrier for the active ingredient.
Description
SUMMARY OF THE INVENTION
This invention relates to a new anticancer antibiotic and a process for the
fermentative production of this new anticancer antibiotic.
More particularly, this invention relates to a new anticancer antibiotic,
now nominated as MI43-37F11 substance. This invention also relates to a
process for the fermentative production of the new anticancer antibiotic,
MI43-37F11 substance. This invention further includes a pharmaceutical
composition comprising MI43-37F11 substance as active ingredient, in
combination with a carrier for the active ingredient.
This invention also relates to a pharmaceutical use of the new antibiotic,
MI43-37F11 substance as an anticancer or cancerocidal agent or as an agent
for enhancement of the production of interleukin-1 in vivo or as an agent
for activation of macrophages in vivo in a mammalian.
BACKGROUND OF THE INVENTION
It is known that many of the known antibiotic substances which are produced
by the cultivation of different kinds of microorganisms can exhibit an
anticancer or antitumor activity. Up to date, some of the known anticancer
antibiotic or antitumor antibiotic substances have extensively been used
as a chemotherapeutic anticancer or antitumor agent and provide an
important mean for therapeutic treatment of cancers or tumors in the
clinic practice. However, many of the known anticancer antibiotic
substances which have been used in the clinic practice can exhibit
considerable toxicity to human body so that the practical use of them is
greatly limited. In this sense, all the known anticancer antibiotics which
have been used in the clinic practice are not necessarily a completely
satisfactory anticancer agent or antitumor agent. Accordingly, there now
remains an outstanding demand to discover and provide such novel
substances that have a low toxicity to human but a high anticancer or
antitumor activity and that can be effectively and safely used for the
therapeutic treatment of a cancer or tumor in human patients. An object of
this invention is to provide a new antibiotic substance which is useful as
an anticancer agent or antitumor agent having the above-mentioned
desirable properties. Another object of this invention is to provide a new
anticancer antibiotic, now nominated as MI43-37F11 substance, which is
utilizable as the anticancer or cancerocidal agent or as the antitumor
agent having the above-mentioned desirable properties in the clinic
practice. A further object of this invention is to provide a process for
the fermentative production of the new anticancer antibiotic, MI43-37F11
substance. Still another objects of this invention will be clear from the
following descriptions.
Thus, we, the present inventors, have made extensive researches in an
attempt to discover and provide a new anticancer antibiotic substance, and
we have now succeeded in obtaining a new anticancer antibiotic, now
nominated as MI43-37F11 substance, and having the following formula [I]:
##STR2##
from the culture of a certain new microbial strain, and have found that
this MI43-37F11 substance exhibits a highly useful anticancer activity
without showing any observable toxicity to mammalian. We have now thus
accomplished this invention.
DETAILED DESCRIPTION OF THE INVENTION
In a first aspect of this invention, therefore, there is provided a new
antibiotic, MI43-37F11 substance which exhibits an anticancer activity and
which is a compound having the following formula [I]:
##STR3##
The new antibiotic, MI43-37F11 substance according to this invention is
remarkedly characterized in that it can exhibit a high activity against
Ehrlich carcinoma.
Physicochemical and biological properties of the antibiotic, MI43-37F11
substance according to this invention are described below.
(A) Physicochemical properties of the antibiotic MI43-37F11 substance
(1) Appearance : Colorless needles.
(2) Molecular weight (as measured by FD mass spectrometry), m/z: 222.
(3) Elementary analysis : Found: C, 59.40%, H, 4.54%, 0, 35.79%.
(4) Empirical formula : C.sub.11 H.sub.10 O.sub.5.
(5) Specific optical rotation : [.alpha.].sub.D 0.degree. C. (c 0.2,
acetonitrile).
(6) Melting point : 148.5-149.5.degree. C.
(7) Ultra-violet absorption spectrum (as shown in FIG. 1 of the
accompanying drawings) :
.lambda..sub.max.sup.methanol 238 nm (4.63), 244 nm (4.65), 256 nm (sh,
4.05), 274 nm (sh, 3.82), 286 nm (sh, 3.67), 330 nm (3.78) (the numerical
data given in the brackets are of the corresponding log s value)
(8) Infrared absorption spectrum (as shown in FIG. 2 of the accompanying
drawing) : characteristic peaks at 3480, 1680, 1630, 1570, 1510, 1230,
1190, 1170, 1100, 10 1090, 980, 860, 840, 710, 690 (cm.sup.-1)
(9) Solubility : easily soluble in acetonitrile, soluble in methanol and
chloroform, but hardly soluble in water and hexane.
(10) Proton nuclear magnetic resonance absorption spectrum : .sup.1 H-NMR
as measured in deutero-acetonitrile is shown in FIG. 3 of the accompanying
drawings (Abscissa : ppm). Tetramethylsilane was used as the internal
standard.
Besides, .sup.13 C-NMR of the MI43-37F11 substance as measured in
deutero-acetonitrile is shown in Table 1 below.
TABLE 1
______________________________________
168.0 s 167.0 s 164.4 s
157.9 s 140.4 s 104.2 d
102.6 d 101.5 d 101.0 s
61.1 t 56.7 q
______________________________________
In Table 1, Note;
s: singlet
d: doublet
t: triplet
q: quartet
Tetramethylsilane was used as the internal standard.
With reference to the accompanying drawings: FIG. 1 is a UV absorption
spectrum of the MI43-37F11 substance according to this invention: FIG. 2
is an IR absorption spectrum of the MI43-37F11 substance according to this
invention : and FIG. 3 is a .sup.1 H-NMR absorption spectrum of the
MI43-37F11 substance according to this invention.
Based on the various physico-chemical properties of the MI43-37F11
substance as described above, the determination of the chemical structure
of the MI43-37F11 substance was made as follows. This substance shows a
molecular ion peak at 222 (m/z) in the FD Mass Spectrometry. The result of
the elementary analysis supports an empirical formula of C.sub.11 H.sub.10
O.sub.5 for this substance. The ultra-violet absorption spectrum as shown
in FIG. 1 and infrared absorption spectrum as shown in FIG. 2 reveal that
the MI43-37F11 substance has an isocumarin skeleton. .sup.1 H-NMR spectrum
as shown in FIG. 3 suggests the existence of the group
##STR4##
and the group
##STR5##
as well as the signals of three aromatic hydrogen atoms and the existence
of one hydroxyl group which is forming the hydrogen-bond. .sup.13 C-NMR
spectrum exhibits eleven signals as shown in the Table 1. The positions of
the substituents CH.sub.2 OH, OCH.sub.3 and OH on the isocumarin nucleus
are decided by comparing with the spectra of Heteronuclear multiple bond
connectivity (A. Bax et al, "J. Am. Soc", 108, 8056-8063 (1986)).
Through the examination of the above-mentioned different spectra of the
MI43-37F11 substance, it has been decided that the MI43-37F11 substance
has the chemical structure of the formula [I]as shown above. The
antibiotic MI43-37F11 substance has also been confirmed to be a novel
antibiotic as there is not reported any known substance which is
coincident with the structure of the formula [I]shown above.
The antibiotic MI43-37F11 substance according to this invention is such a
compound which is found at the first time to exhibit the anticancer
activity, amongst the known isocumarins. Besides, owing to its low
toxicity to mammalian, the antibiotic MI43-37F11 substance is expectable
to be an anticancer antibiotic useful in the chemotherapeutic treatment of
cancer-bearing patients.
By the way, we have been aware of that Japanese patent application
publication "Kokai" No. 71076/78 (published on 24 June 1978) discloses
3-hydroxymethyl-6, 7-dimethoxy-8-hydroxyisocumarin of formula
##STR6##
and 3-hydroxymethyl-6,8-dihydroxy-7-methoxyisocumarin having an inhibitory
activity to cyclic adenosine monophosphate (CAMP) phosphodiesterase. In
said Japanese patent application publication, there is described that the
isocumarin derivatives as disclosed therein would be expectable to exhibit
an anticancer activity, a hypotensive activity, an anti-inflammatory
activity and anti-allergic activity etc., but there is not given at all
any experimental data for showing that the isocumarin derivatives as
disclosed therein actually can exhibit any anticancer activity.
(B) Biological properties of the antibiotic MI43-37F11 substance
(1) Antitumor activity of MI43-37F11 substance to inhibit the proliferation
of various sorts of experimental tumor cells and human cancer cells.
A cell suspension containing cells of mouse Leukemia L1210, mouse Leukemia
P388, mouse Leukemia EL4, mouse IMC carcinoma or human lung cancer LX-1
cells in a culture medium containing 10% calf serum at a cell density of 1
x 105 cells/ml was prepared, and 200 .mu.l-portion of the cell suspension
so prepared was placed in a microplate and then added with a solution of
MI43-37F11 substance at varying concentrations (dissolved in a mixture of
water and dimethylsulfoxide (95:5)). The cell suspension containing the
test compound added was subsequently incubated at 37.degree. C for 2 days
(but 5 days for the LX-1 cells).
After the incubation, the number of the cells of the L1210 cells, P388
cells, the EL4 cells and the IMC carcinoma cells in the treated groups was
counted by means of Coulter-counter. The control group (untreated) of the
cell suspension to which the test compound was not added was incubated in
the same way as above and then the number of the cells was similarly
counted.
Rate (%) of inhibition of the cell proliferation of the treated group
against that of the control group (untreated) was evaluated.
With the human lung cancer LX-1 cell, however, the incubated cell
suspension of LX-1 cells was added with 10 .mu.l of a solution containing
5 mg/m(of MTT (namely, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide), followed by further incubation at 37.degree. C. for
4 hours. After the completed incubation, the supernatant liquor (100
.mu.l) was removed from the incubated cell suspension and the resultant
residual suspension containing the LX-1 cells was added with 150 .mu.l of
a mixture of isopropanol and lN-HC((25:1). The resulting mixture was
evaluated for its absorption of light ray at 540 nm. Rate (%) of
inhibition of the proliferation of the LX-1 cells in the treated group
against that of the control group (untreated) was evaluated in terms of
the evaluated values of the absorption of light ray at 540 nm.
From the values of the rate (%) of inhibition as evaluated as above, there
were estimated the values of IC.sub.50 (.mu.g/m;) of MI43-37F11 substance
of this invention, that is, the concentration of MI43-37F11 substance
which can give 50% inhibition of the proliferation of the various cancer
cells tested. The test results are summarized in Table 2 below.
TABLE 2
______________________________________
IC.sub.50 Value
Tested cells (.mu.g/ml)
______________________________________
Mouse leukemia L1210
50
Mouse leukemia P388
50
Mouse leukemia EL4 52
Mouse IMC carcinoma
72
Human lung cancer LX-1
100
______________________________________
(2) Antitumor effect of the antibiotic MI43-37F11 substance on Ehrlich
carcinoma was estimated by the following procedures
(a) Experiments for therapeutic treatment of Ehrlich carcinoma-bearing mice
were conducted by intraperitoneal injection of the antibiotic MI43-37F11
substance. Thus, a cell suspension of Ehrlich ascites carcinoma cells was
subcutaneously inoculated at the abdominal region of ICR mice (female,
6-week old, 4 mice in each treated group) so that the number of the
carcinoma cells as subcutaneously transplanted was 2.times.10.sup.6 cells
per mouse. After the transplantation of the carcinoma cells, the
antibiotic MI43-37F11 substance was administered by intraperitoneal
injection to the mice having the transplanted and firmly grown carcinoma
cells, according to the following schedule of administration. Namely,
MI43-37F11 substance was administered at a dosage of 10 mg/kg or 2.5 mg/kg
or 0.625 mg/kg only once on the 7th day after the transplantation of the
cells, or alternatively MI43-37F11 substance was administered at a dosage
of 2.5 mg/kg or 1.25 mg/kg or 0.625 mg/kg, totally five times but every
other day from the 7th day to the 14th day after the transplantation of
the carcinoma cells. The administration of MI43-37F11 substance was not
made but intraperitoneal injection of physiological saline was made in the
mice of the control group (untreated) (8 mice in each control group).
On the 15th day after the transplantation of the carcinoma cells, the mass
of the tumor (carcinoma) was surgically removed out of each mouse and
weighed. The rate of the reduction in the weight of the tumor (carcinoma)
obtained from the group of mice treated with MI43-37F11 substance was
evaluated in term of the rate (%) of inhibition, with such an assumption
that the weight of the tumor obtained from the control group of mice was
amounting to 100. In other words, the rate (%) of inhibition to the growth
of Ehrlich carcinoma was calculated according to the following equation
##EQU1##
where C means the weight of the tumor in the control group of mice
(untreated) and T means the weight of the tumor of the treated group of
mice.
The test results obtained are shown in Table 3 below.
TABLE 3
______________________________________
Dosage of Rate (%) of Inhibition
MI43-37F11 Schedule of administration
Substance 7th day 7th day to 14th day (every
(mg/kg/day) (only once)
second day, totally 5 times)
______________________________________
10 59 --
2.5 51 60
1.25 -- 67
0.625 12 30
______________________________________
(b) Another experiments for therapeutic treatment of Ehrlich
carcinoma-bearing mice were conducted by oral administration of MI43-37F11
substance. Thus, the cell suspension of Ehrlich ascites carcinoma cells
was subcutaneously inoculated at the abdominal region of ICR mice (female,
6-week old, 5 mice in each treated group) so that the number of the
carcinoma cells as subcutaneously implanted was 2.times.10.sup.6 cells per
mouse. The MI43-37F11 substance was orally administered to the mice of the
treated group at a dosage of 6.3 mg/kg, 12.5 mg/kg, 25 mg/kg, 50 kg/mg or
100 mg/kg once a day during the consecutive 9 days from the 1st day to the
9th day after the implantation of the carcinoma cells. On the 14th day
after the implantation of the carcinoma cells, the mass of the tumor
(carcinoma) was surgically removed out of each mouse and weighed. The
weight of the tumor obtained from the treated group of mice was compared
with the weight of the tumor obtained from the control group of mice
(untreated), by calculating the rate (%) of inhibition according to the
same equation as above.
The test results obtained are shown in Table 4 below.
TABLE 4
______________________________________
Dosage of MI43-37F11
Weight of tumor
Rate (%) of
substance (mg/kg/day)
(mg .+-. S.D.)
Inhibition
______________________________________
0 1,084 .+-. 256
--
6.3 586 .+-. 282*
45
12.5 391 .+-. 266**
63
25.0 280 .+-. 152***
74
50.0 485 .+-. 123**
55
100.0 566 .+-. 421**
47
______________________________________
***P < 0.001, **P < 0.01, *P < 0.05
(3) Experiments for estimation of the activity of MI43-37F11 substance to
activate peritoneal macrophages were conducted by the following procedure.
Thus, one day before the harvest of the peritoneal macrophages, the
antibiotic MI43-37F11 substance was intraperitoneally administered to
CDF.sub.1 mice (8-week old, female, 3 mice in each group) at a dosage of
50 mg/kg, 12.5 mg/kg or 3.125 mg/kg. One day after the administration, the
macrophages were harvested from the peritoneal cavity of the treated mice
and placed into a culture medium present in a plastic dish (Falcone, No.
3002, 60 x 15 cm, a product of Decton Dikinson & Company) at the cell
concentration of 1.times.10.sup.6 cells/m(, followed by incubation of the
macrophages. The incubated medium containing the macrophages was then
added with 100 ng/ml of phorbol myristate acetate to elicite the
macrophages for their production of O.sub.2 -. The amount of O.sub.2 - was
determined by reductive reaction with cytochrome C. The control group of
mice (untreated) received only intraperitoneal injection of physiological
saline in place of the administration of MI43-37F11 substance before the
harvest of the peritoneal macrophages was done. Assumed that the amount of
O.sub.2 - as produced and liberated by the peritoneal macrophages which
were harvested from the mice of the control group was amounting to 100,
the rate of increase in the amount of O.sub.2 - as produced by the
peritoneal macrophages of the mice of the treated group was evaluated. In
other words, the rate (%) of increase in the production of O.sub.2 - by
the peritoneal macrophages of the mice of the treated group was calculated
according to the following equation;
##EQU2##
where T means the amount of O.sub.2.sup.- produced by the peritoneal
macrophages of the mice of the treated group and C means the amount of
O.sub.2.sup.- produced by the peritoneal macrophages of the control group
(untreated).
The test results obtained are summarized in Table 5 below.
TABLE 5
______________________________________
Dosage of MI43-37F11
Rate (%) of increase
substance (mg/kg)
in production of O.sub.2.sup.-
______________________________________
50 255
12.5 127
3.125 105
______________________________________
(4) Experiments for estimation of the effect of the antibiotic MI43-37F11
substance on phagocytosis of the peritoneal macrophages were conducted by
the following procedure. Thus, 3 days and one day before the harvest of
the peritoneal macrophages, MI43-37F11 substance was intraperitoneally
administered to CDF.sub.1 mice (female, 8-weeks old, 3 mice) at a dosage
of 50 mg/kg, 5 mg/kg or 0.5 mg/kg. One day after the administration of
MI43-37F11 substance, the peritoneal macrophages were harvested from the
peritoneal cavity of the mice of the treated group of mice and then
inoculated into a culture medium present in a plastic dish (Falcone) at
the cell density of 5.times.10.sup.5 cells/m(, followed by incubation of
the macrophages. The incubated medium containing the macrophages was then
added with 7.5.times.10.sup.6 cells/ml of heat-treated yeast cells,
followed by further incubation for a time. The macrophages were then fixed
by addition of methanol and stained by Giemsa staining method. The number
of the yeast cells which received the phagocytosis by 400 macrophages was
counted. The control group of mice (untreated) received only
intraperitoneal injection of physiological saline in place of the
administration of MI43-37F11 substance, before the harvest of the
peritoneal macrophages was done. Assumed that the number of the yeast
cells which had received the phagocytosis by 400 macrophages as harvested
from the mice of the control group was amounting to 100, the rate of
increase in the phagocytic action of the peritoneal macrophages as
harvested from the treated group of mice was evaluated. In other words,
the rate (%) of increase in the phagocytic action of the peritoneal
macrophages of the mice of the treated group was calculated according to
the following equation:
##EQU3##
where T means the mumber of yeast cells having received the phagocytosis
by 400 macrophages which were harvested from the treated group of mice,
and C means the number of yeast cells having received the phagocytosis by
400 macrophages which were harvested from the control group of mice
(untreated).
The test results are shown in Table 6 below.
TABLE 6
______________________________________
Rate (%) of Increase in
Dosage of MI43-37F11
phagocytic action of
substance (mg/kg)
peritoneal macrophages
______________________________________
50 144
5 80
0.5 102
______________________________________
(5) Experiments for estimation of the activity of the antibiotic MI43-37F11
substance to enhance the production of interleukin-1 in vivo was conducted
by the following procedure. Thus, MI43-37F11 substance was orally
administered to CDF.sub.1 mice (female, 6-week old) B at a dosage of 25
mg/kg. One day, 3 days, 5 days and days after the administration,
peritoneal exudate cells (PEC) and spleen cells were respectively
harvested from the mice of the treated group. The peritoneal exudate cells
and spleen cells as harvested were independently divided into two classes,
namely the class of the adherent cell which was adherent in its nature to
the wall of a plastic dish and the class of the non-adherent cell. The
whole peritoneal exudate cells (PEC), the adherent cells of PEC and the
adherent cells of the spleen cell were separately suspended into aliquots
of a culture medium to give a cell concentrations of 1.times.10.sup.6
cells/well, 2.times.10.sup.6 cells/well and 2.5.times.10.sup.6 cells/well,
respectively. After the incubation for 24 hours, the supernatants were
collected by filtration of the incubated culture media and then assayed
for their activity of interleukin-1 (IL-1) according to the method of
Jonathan K. et al (see "Lymphokine Research" 3 (4), 175-182, (1984)).
Namely, 100 .mu.l of each supernatant obtained as above and Concanavalin A
(to a final concentration of 2.5 .mu.g/ml) were added to 100 .mu.l of a
cell suspension containing 1 x 105 cells/m(of D10.G4.1. cells which will
proliferate in the presence of Concanavalin A plus interleukin-1 when the
latter compounds are given. The resulting mixture was then incubated for
48 hours and then added with .sup.3 H-thymidine (.sup.3 H-TdR), followed
by further incubation for 16 hours. After this, the rate of uptake of
.sup.3 H-TdR by the D10.G4.1. cells was determined in term of the
radio-activity unit of c.p.m. The rate of uptake of .sup.3 H-TdR in the
control group of mice (untreated) was also determined in the same manner
as above.
The test results obtained are summarized in Table 7 below.
TABLE 7
__________________________________________________________________________
Days after Uptake of .sup.3 H-TdR (c.p.m.)
oral administration of
Whole peritoneal
Adherent cell Adherent cell
MI43-37F11 substance
exudate cells (PEC)
in PEC in spleen cells
(25 mg/kg) c.p.m. .+-. S.D.
T/C %
c.p.m. .+-. S.D.
T/C %
c.p.m. .+-. S.D.
T/C %
__________________________________________________________________________
Control 7166 .+-. 752
100 10561 .+-. 1241
100 2091 .+-. 481
100
1 day 4393 .+-. 397**
61.3
17510 .+-. 810**
165.8
1741 .+-. 352
83.3
3 days 5988 .+-. 602
83.6
13912 .+-. 1367**
131.7
4868 .+-. 497**
232.8
5 days 28072 .+-. 1174***
391.7
26759 .+-. 3601***
253.4
2140 .+-. 121
102.3
7 days 3906 .+-. 465**
54.5
17639 .+-. 2699*
167.0
2385 .+-. 134
114.1
__________________________________________________________________________
In a second aspect of this invention, there is provided a process for the
production of the anti-cancer antibiotic, MI43-37F11 substance having the
formula [I] as described hereinbefore, which comprises culturing an
MI43-37F11 substance-producing strain of the genus Streptoverticillium in
a culture medium containing assimilable carbon sources and assimilable
nitrogen sources, until a substantial amount of MI43-37F11 substance is
produced and accumulated in the culture, and then recovering MI43-37F11
substance having the above formula [I]from the resulting culture.
One typical example of the MI43-37F11 substance-producing strain available
in the process of the second aspect of this invention is a new strain
designated as MI43-37F11 strain, which belongs to the genus
Streptoverticillum, and which was isolated by us from a soil sample
collected in Midori-ku, Yokohama City, Kanagawa Prefecture, Japan. This
MI43-37F11 strain has the following microbiological properties.
1. Morphological observations
Microscopic observation shows that the MI43-37F11 strain has branched
substrate mycelia, from which aerial hyphae develop with formation of
whirls. It is observed that a chain of more than 10 spores is formed at
the tip of the aerial hyphae. No spiral-formation is observed. Spores are
measuring about 0.7-0.8 by 1.1-1.3 microns in size annd have smooth
surface.
2. Growth charcteristics on various culture media
The standard given in each of the brackets [ ] for the description of color
is according to "Color Harmony Manual" of Container Corporation of
America.
(1) Sucrose-nitrate-agar medium (cultured at 27.degree. C.)
White aerial hyphae are thinnly formed on the colorless growth. No soluble
pigment is observed.
(2) Glucose-asparagine-agar medium (cultured at 27.degree. C.)
Aerial hyphae of yellow-tinged white [11/2db, Parchmeat] in color are
thinnly formed on the growth of colorless to pale yellow [2ea, Lt Wheat]
to pale yellowish brown [2 gc, Bamboo]in color. White cottonyl aerial
hyphae are also partly formed. No soluble pigment is observed.
(3) Glycerine-asparagine-agar medium (ISP-medium 5, cultured at 27.degree.
C.)
10 Colorless aerial hyphae are formed on the growth of colorless to pale
yellowish brown [2 ie, Lt Mustard Tan] in color. The soluble pigment is
tinged with brown.
(4) Starch-inorganic salt-agar medium (ISP-medium 4, cultured at 27.degree.
C.)
Aerial hyphae of yellowish white [111/2ca, Cream] to bright olive gray
[11/2 ge, Lt Olive Gray] in color are formed on the growth of colorless to
pale yellowish brown [2 le, Mustard - 2 ne, Mustard Gold]in color. No
soluble pigment is observed.
(5) Tyrosine-agar medium (ISP-medium 7, cultured at 27.degree. C.)
Aerial hyphae of white to brownish white [3 ba, Pearl--2 cb, Ivory Tint]in
color are formed on the growth of pale yellowish brown [2 ie, Lt Mustard
Tan] to bright grayish brown [2 lg, Mustard Tan]in color. No soluble
pigment is observed.
(6) Nutrient agar medium (cultured at 27.degree. C.)
White aerial hyphae are thinnly formed on the growth of colorless to pale
yellowish brown [2 ie, Lt Mustard Tan] in color. Black-tinged soluble
pigment is slightly produced.
(7) Yeast-malt agar medium (ISP-medium 2, cultured at 27.degree. C.)
Aerial hyphae of white to brownish white [3 ba, Pearl] in color are formed
on the wrinkled growth of colorless to pale yellowish brown [2 ic, Honey
Gold --3 ic, Lt Amber] in color. No soluble pigment is observed.
(8) Oatmeal agar medium (ISP-medium 3, cultured at 27.degree. C.)
Aerial hyphae of white to yellowish white are thinnly formed on the growth
of colorless to pale yellow [2 ea, Lt Wheat - 2 ca, Lt Ivory]in color. No
soluble pigment is observed.
(9) Glycerin-nitrate-agar medium (cultured at 27.degree. C.)
Aerial hyphae of white to yellowish white [11/2 db, Parchment] are thinly
formed on the growth of colorless to pale yellow [2 ca, Lt Ivory]in color.
No soluble pigment is observed.
(10) Starch agar medium (cultured at 27.degree. C.)
Aerial hyphae of white to yellowish white [2 ca, Lt Ivory]are formed on the
growth of colorless to pale yellow [2 ea, Lt Wheat]to pale yellowish brown
[11/2 ic, Lt Antique Gold]in color. No soluble pigment is observed.
(11) Calcium malate-agar medium (cultured at 27.degree. C.)
White aerial hyphae are slightly formed on the colorless and very poor
growth. No soluble pigment is observed.
(12) Cellulose (synthetic test solution containing filter paper pieces,
cultured at 27.degree. C.)
The growth is colorless and aerial hyphae are not formed thereon. No
soluble pigment is observed.
(13) Gelatin stab
In a 15% simple gelatin culture medium (cultured at 20.degree. C.), the
growth is colorless but no aerial hyphae are formed, and soluble pigment
of pale yellowish brown is observed. In glucose-peptone-gelatin culture
medium (cultured at 27.degree. C.), the growth is colorless but no aerial
hyphae are formed, and soluble pigment of slightly brownish color is
observed.
(14) Skimmed milk (cultured at 37.degree. C.)
The growth is cololess to pale brown and no aerial hyphae are formed.
Soluble pigment of slightly yellowish to brownish color is observed.
3. Physiological properties
(1) Temperature range for the growing
In the tests which were conducted using a starch-inorganic salt-agar medium
(ISP-medium 4, BACTO-INORGANIC-SALTS STARCH AGAR) and incubating the
MI43-37F11 strain at different temperatures of 20.degree. C., 24.degree.
C., 27.degree. C., 30.degree. C., 37.degree. C. and 50.degree. C., the
MI43-37F11 strain grows at all the temperatures tested, but not at
50.degree. C. Optimum temperature for good growth appears to be in the
vicinity of 27.degree. C.
(2) Liquefaction of gelatin (in 15% simple gelatin medium, cultured at
20.degree. C.; and in glucose-peptone-gelatin medium, cultured at
27.degree. C.)
Liquefaction started about at the third day of the incubation both in the
15% simple gelatin medium and in the glucose-peptone-gelatin medium. The
grade of liquefaction is medium to rather strong.
(3) Hydrolysis of starch (starch-inorganic salt agar medium and starch-agar
medium, each cultured at 27.degree. C.)
Hydrolysis started about at the third day of the incubation both in the
starch-inorganic salt agar medium and in the starch agar medium, where the
grade of hydrolysis is rather strong.
(4) Coagulation and peptonization of skimmed milk (skimmed milk, cultured
at 37.degree. C.)
Coagulation was exhibited about at the second day of the incubation and
then was completed at the third day of the incubation, and thereafter
immediately the peptonization started. The peptonization proceeded slowly
and was not completed even after 3 weeks of the incubation.
(5) Formation of melanoid pigment (Trypton-yeast broth, ISP-medium 1;
peptone-yeast-iron agar medium, ISP-medium 6; tyrosine-agar medium,
ISP-medium 7; each cultured at 27.degree. C.)
The melanoid formation was positive in the peptone-yeast-iron medium and
was apparently positive in the tryptone-yeast-broth but was negative in
the tyrosine-agar medium.
(6) Utilization of various carbon sources (Pridham-Gottlieb agar medium,
ISP-medium 9, cultured at 27.degree. C.)
Glucose, fructose and inositol are utilizable for the growth. But,
L-arabinose, D-xylose, sucrose, rhamnose, raffinose, D-mannitol and
lactose are not utilizable.
(7) Liquefaction of calcium malate (calcium malate agar medium, cultured at
27.degree. C.)
Liquefaction of calcium malate in the calcium malate agar was negative.
(8) Reduction of nitrate (aqueous peptone solution containing 0.1%
potassium nitrate, ISP-medium 8, cultured at 27.degree. C.)
The reduction was rather faintly positive.
(9) Decomposition of cellulose (synthetic test solution containing filter
paper pieces, cultured at 27.degree. C.)
The decomposition of cellulose was negative.
Summarizing the microbiological properties described above, the MI43-37F11
strain is morphologically characterized in that the aerial hyphae have
whirls, but the formation of spirals is not observed. The spore surface is
smooth. The aerial hyphae of white to brown tinged white, or yellowish
white, or occasionally bright olive gray in color are formed on the growth
of colorless to pale yellowish brown color, in various culture media.
Soluble pigment is not produced, or occasionally soluble pigment of brown
tinged color is observed. The formation of the melanoid pigment is
negative in the tyrosine-agar medium, but is positive in the
tryptone-yeast broth and in the peptone-yeast-iron agar medium. The grade
of the protein-decomposing activity is medium to rather strong, and the
grade of the starch-hydrolyzing activity is rather strong, too.
2,6-Diaminopimelic acid present in the cell wall is of the LL-type.
In view of the microbiological properties described above, we have judged
that the MI43-37F11 strain belongs to the genus Streptoverticillium.
When searching analogous known species with reference to the properties of
the MI43-37F11 strain, Streptoverticillium eurocidicum [Literature 1:
"International Journal of Systematic Bacteriology", Vol. 22, p. 293 (1972)
; Literature 2 : ditto, Vol. 30, p. 408 (1980) ; Literature 3 : "The
Journal of Antibiotics, Ser. A.", Vol. 7, p. 98 (1954)]and
Streptoverticillium albireticuli [Literature 1 : "International Journal of
Systematic Bacteriology", Vol. 18, p. 80 (1968) ; Literature 2 : ditto,
Vol. 30, p. 407 (1980)]appear to resemble to the MI43-37F11 strain.
Then, our study was made for the comprisons between the properties of the
MI43-37F11 strain, and the properties of Streptoverticillium eurocidium
and of Streptoverticillium albireticuli as described in the above
literatures. The results of these comprisons are summarized in the Table 8
below.
TABLE 8
__________________________________________________________________________
Streptoverticillium
Streptoverticillium
eurocidicum albireticuli
MI43-37F11
IMC S-0770(ISP5604)
IMC S-0222(ISP5051)
__________________________________________________________________________
Nature of aerial hyphae
Formation of
Formation of
Formation of
whirls whirls whirls
Spore surface
Smooth Smooth Smooth
Color of aerial hyphae
White to yellow
White to yellow
White to yellow
tinged white
tinged white
tinged white
brownish white,
brownish white,
or bright olive
or bright olive
gray gray
Color of growth
Colorless to
Colorless to
Colorless to
pale yellowish
pale yellowish
pale yellowish
brown brown brown
Soluble pigment
-- -- --
Formation of melanoids Lit.*1
Lit.*2 Lit.*3
Lit.*4
In ISP-medium 1
(+) + + + No
description
In ISP-medium 6
+ + + + +
In ISP-medium 7
.-+. .-+.
- ? + +
Hydrolysis of starch
+ + + + + + + + +
+ + +
Coagulation of
+ + + + + + - + + +
skimmed milk
Peptonization of
+ (+) - .-+. slow
skimmed milk
Gelatin Liquefaction
In 15% simple
+ + .-+. - (+) slow
gelatin medium slow
In glucose-peptone-
+ + + + +
gelatin medium
Reduction of nitrate
(+) + - + +
Carbon source
utilization*
D-glucose + + + + +
L-arabinose - - - - -
D-xylose - - - - -
D-fructose (+) (+) + .+-. ?
Sucrose - - - - -
Inositol + + + + +
Rhamnose - - - - -
Raffinose - - - - -
D-mannitol - - - - -
Lactose - - -
__________________________________________________________________________
Note:
In Table 8, the symbol ".-+." means "possibly (-)", "?" means "doubtfully
and "(+)" means "possibly +".
*Particularly for carbon source utilization, the symbol "+" means
"utilizable", "(+)" means "possibly utilizable"; ".+-." means "doubtfully
either "+" or "-", "?" means "variable", and "-" means "not utilizable".
Literature*1 "International Journal of Systematic Bacteriology", Vol. 22,
p. 293 (1972)
Literature*2 "The Journal of Antibiotics Ser. A", Vol. 7, p. 98 (1954)
Literature*3 "International Journal of Systematic Bacteriology", Vol. 18,
p. 80 (1968)
Literature*4 S. A. Waksman; "The Actinomycetes", Vol. 2, p. 169 (1961).
As can be seen from Table 8 above, the MI43-37F11 strain closely resembles
both to Streptoverticillum eurocidicum and Streptoverticillium
albireticuli. However, in view of the bright olive gray color of the
aerial hyphae, the probably "negative" formation of melanoid pigment in
the ISP-medium 7, probable utilization of D-fructose and other properties
of the MI43-37F11 strain, it is judged that the MI43-37F11 strain is most
closely analogous to Streptoverticillium eurocidicum. Thus, the MI43-37F11
strain has now been identified as Streptoverticillium eurocidicum
MI43-37F11 .
The strain MI43-37F11 has been deposited in the Japanese depository
"Fermentation Research Institute", Agency of Industrial Science and
Technology (located at Tsukuba-City, Ibaraki Prefecture, Japan), since
Jan. 27, 1989 under the deposit number "FERM P-10513" and now deposited
under the deposit number "FERM BP-2783" in terms of the Budapest Treaty.
In carrying out the process for the production of the anticancer antibiotic
MI43-37F11 substance according to the second aspect of this invention, an
MI43-37F11 substance-producing strain belonging to the genus
Streptoverticillium is cultivated by a known and ordinary method for the
cultivation of microorganisms of Actinomyces. Thus, an amount of the
MI43-37F11 substance-producing strain is inoculated to a suitable culture
medium comprising assmilable carbon and nitrogen sources and is then
incubated under aerobic conditions, preferably under submerged aerobic
conditions, so that the MI43-37F11 substance is produced and accumulated
in the culture broth.
The culture medium used for the cultivation may contain corbon sources,
nitrogen sources and inorganic salt, etc., which are customarily used for
the cultivation of Actinomyces. The nitrogen sources include those
commercially available known materials such as peptone, meat extract, corn
steep liquor, cotton seed meal, peanut powder, soybean powder, yeast
extract, NZ-amine, casein-hydrolysate, sodium nitrate, ammonium nitrate
and ammonium sulfate. The carbon sources include those commercially
available known materials, for example, carbohydrate such as glycerin,
starch, glucose, galactose, mannose and mollasses, and fats and oils. The
inorganic salts may include sodium chloride, phosphates, calcium
carbonate, magnesium sulfate etc.
The productive culture medium which may be used for commercial production
of MI43-37F11 substance may contain a slight amount of one or more
inorganic salts, and also may contain as anti-foaming agent animal oils,
vegetable oils and mineral oils. Further, any other organic and inorganic
materials which are known as the material useful for potentiation of the
microorganisms and useful for enhanced production of MI43-37F11 substance
may also be advantageously employed as the additive in the culture medium.
The cultivation of the MI43-37F11 strain for the commercial production of
MI43-37F11 substance may be conducted under submerged, aerobic conditions
and the cultivation temperature may be in a range of the temperature where
the MI43-37F11 substance-producing strain can grow and produce a
substantial amount of the MI43-37F11 substance, usually in a range of from
27.degree. C. to 37.degree. C. The other conditions for the cultivation
may be selected, depending on and, according to the microbiological and
physiological properties of the MI43-37F11 substance-producing strains.
The recovery of the MI43-37F11 substance from the culture of the MI43-37F11
substance-producing strain may be achieved in the following manner. The
antibiotic MI43-37F11 is mainly accumulated in the liquid phase of the
culture broth. Thus, the culture broth obtained is filtered, and the broth
filtrate is adjusted to faintly acidic pH and extracted with a
water-immiscible organic solvent such as ethyl acetate. In addition to the
extraction method described above, any conventional methods for isolation
of oleophilic substances, namely adsorption chromatography, gel-filtration
chromatography, high performance liquid chromatography, and a combination
thereof may also advantageously be employed for the isolation and
purification of the MI43-37F11 substance.
This invention also includes use of the MI43-37F11 substance in a
pharmaceutical composition.
On the basis of such useful biological properties of the MI43-37F11
substance as given above, a third aspect of this invention is a
pharmaceutical composition comprising as active ingredient the antibiotic,
MI43-37 F11 substance having formula (I) above, in combination with a
pharmaceutically acceptable carrier or carriers for the active ingredient.
The pharmaceutical composition according to this invention is effective
and useful as carcinostatic or antitumor agent for mammalian animals,
including man.
The pharmaceutical composition according to this invention may be
formulated in a conventional manner into any convenient form of medicinal
preparations for oral, intraperitoneal or parenteral administration such
as, for example, injections, tablets, capsules, granules, syrups,
suppositories and ointments. As pharmaceutically acceptable carriers,
there may be used any of known, conventional ones as desired. The nature
and composition of carreirs to be used may vary depending on the route and
manner of administration and include organic and inorganic, solid and
liquid, usually inert carriers and excipients known and available for
pharmaceutical purposes. Some concrete examples of such carriers are
crystalline cellulose, gelatin, lactose, starch, magnesium stearate, talc,
vegetable and animal fats and oils, gums and polyalkylene glycols among
others. The concentration of the active carcinostatic or antitumor
ingredient, MI43-37F11 substance, in the pharmaceutical composition of
this invention may vary from 0.2 to 100% by weight, preferably from 1 to
90% by weight, based on the total weight of the composition. If desired,
the pharmaceutical composition of this invention may contain, in addition
to the MI43-37F11 substance, one or more other pharmacologically active
ingredients including those having carcinostatic, antitumor and other
pharmacological activities.
The pharmaceutical composition according to this invention may be
administered at a dosage capable of exhibiting a desired pharmacological
activity without being accompanying with any appreciable side effect.
Particular dosage is to be chosen by medical expert in each particular
case, but the dosage of the active ingredient, the MI43-37F11 substance
will, in general, be a level in the range of 10 mg-10 g, preferably 20 mg
- 5 g, per day on adult patient for therapeutic treatments of carcinomas
and malignant tumors. In these cases, the pharmaceutical composition of
this invention may conveniently be administered as a unit preparation
containing 1 m-5 g, preferably 3 mg-1 g of the active ingredient,
MI43-37F11 substance.
Thus, according to a fourth aspect of this invention, there is provided a
method of inhibitingly treating carcinomas or malignant tumor of mammalian
animals, including man, which comprises administering the MI43-37F11
substance having formula (I) above, usually in the form of a
pharmaceutical composition, in a therapeutically effective amount to a
mammalian animal having a carcinoma or tumor.
As already mentioned briefly, the dosage of the MI43-37F11 substance may
suitably be determined by medical experts typically having regard to the
age, body weight, symptom of patients and therapeutic purpose as intended.
The effective dosage as indicated above can be administered continuously
or intermittently as long as the total dosage does not exceed such a
specific level as decided in view of results of animal tests and various
circumstances.
Furthermore, still another aspect of this invention include pharmaceutical
use of the antibiotic, MI43-37F11 substance of the formula (I) as defined
hereinbefore, as an anticancer or cancerocidal agent or as antitumor agent
or in the manufacture thereof. This invention also include pharmaceutical
use of the MI43-37F11 substance as an agent for enhancement of the
production of interleukin-1 in vivo in a mammalian, including man, or in
the manufacture of said agent. These agents may be in the form of a
pharmaceutical composition comprising the MI43-37F11 substance as active
ingredient, in association with a pharmaceutically acceptable carrier for
the active ingredient.
Besides, as described hereinbefore, the MI43-37-F11 substance of this
invention is effective to enable the peritoneal macrophages to make their
increased production of 02- and also effective to enhance the phagocytic
action of the macrophages as well as the in vivo production of
interleukin-1, so that the MI43-37F11 substance of this invention is
virtually able to enhance the activities of macrophages in vivo in a
mammalian. Accordingly, a further aspect of this invention includes a use
of the MI43-37F11 substance as an agent for activation of macrophage in
vivo in a mammalian.
This invention is now illustrated with reference to the following Examples,
to which this invention is not limited in any way.
EXAMPLE 1
A loopful amount of Streptoverticillium eurocidicum MI43-37F11 strain
(identified as FERM BP-2783) was taken from its agar slant culture and
then inoculated into 3 Elemmyer flasks which had each contained 110 ml of
a sterilized culture medium comprising 2.0% galactose, 2.0% dextrin, 1.0%
soy-pepton ("Bacto Soyton", a product of Difco Co., Ltd.), 0.5% corn steep
liquor (a product of Nihon Shokuhin Kako Co., Ltd.), 0.2% ammonium
sulfate, 0.2% calcium carbonate and 0.003% antifoaming agent, silicon oil
"Silicon KM70" (a trade name of a product of Shinetsu Chemicals Co.,
Ltd.), as adjusted to pH 7.0. The inoculated culture media were then
incubated at 30.degree. C for 2 days under agitation, so that a seed
culture was prepared.
2.5 ml -portions of the seed culture so obtained were each inoculated into
80 Sakaguchi flasks which had each contained 125 ml of a culture medium
comprising 2.0% glycerine, 1.5% "Esusan Meat" (a trade name of a soybean
flour product of Ajinomoto Co., Ltd.), 0.1% potassium hydrogen phosphate,
0.0005% cobalt chloride 6-hydrate and 0.003% antifoaming silicone, as
adjusted to pH 6.2 with dipotassium phosphate. The cultivation of the
MI43-37F11 strain was then conducted at 28.degree. C. for 4 days. The
resulting culture broth was filtered to remove the mycelia, and the
recovered broth filtrate (adjusted to pH 5) was extracted with an equal
volume of ethyl acetate. The resulting extract in ethyl acetate was
concentrated under a reduced pressure to yield a brownish oily substance
(2.0 g) containing the MI43-37F11 substance. 10 This crude product of the
MI43-37F11 substance was mixed with 10 g of silica gel, followed by drying
under reduced pressure. The resulting dried mixture was placed on the top
of a column of 60 m(of silica gel, followed by carrying out a silica gel
column chromatography. The silica gel column was previously packed with
the silica gel with aid of n-hexane. The silica gel column having said
mixture on the top thereof was first washed with a mixture (300 m() of
n-hexane and ethyl acetate (8:2, V/V) and then eluted with a mixed solvent
of n-hexane and ethyl acetate (1:1, V/V). The resulting eluate was
concentrated under a reduced pressure to afford a brown colored oily
substance (0.2 g) containing the MI43-37F11 substance.
This oily substance was mixed with 2 g of silica gel, followed by drying
under reduced pressure. The resulting dried mixture was placed on the top
of a column of 20 ml of a silica gel which had been packed into the column
with aid of n-hexane. After this, for the column chromatography, the
silica gel column having said mixture at the top thereof was first washed
with 100 ml of a mixture of n-hexane-ethyl acetate(7:3, V/V) and then
eluted with 100 m(of a mixture of n-hexane-ethyl acetate (6:4, V/V). The
elute from the silica gel column was collected in 5 m(-fractions. The
active fractions containing the MI43-37F11 substance were combined and
concentrated under a reduced pressure to give 115 mg of a yellow colored
oily substance.
This yellow oil product was dissolved in a small volume of acetonitrile and
the solution was subjected to a high performance liquid chromatography on
a reverse phase column (20 mm in diameter.times.300 mm in height) of
octadodecyl silanide-coated silica gel (commercially available under a
tradename "Senshu Pack" ODS 330IN, a product of Senshu Kagaku Co., Japan)
in such a way that the elution was effected at a flow rate of 4 m(/minute
with a linear concentration gradient from 20% acetonitrile to 50%
acetonitrile in water. The eluate was collected in 4 m(-fractions. Among
these collected fractions, the active fractions containing the MI43-37F11
substance were combined and concentrated in vacuo to afford 50 mg of a
yellow colored powder comprising the MI43-37F11 substance.
This yellow powdery substance was again subjected to a high performance
liquid chromatography on a silica gel column (20 mm in diameter.times.250
mm in height) of "YMC-Pack, A-043SIL" (a product commercially available
from Yamamura Kagaku Kenkyujo Co., Japan) with a mixed solvent of
n-hexane-chloroform (1:9, V/V) as eluent in such a way that the elution
was effected at a flow rate of 4 m(/minute and the eluate was collected in
4 m(-fractions. The active fractions containing the MI43-37F11 substance
were combined and concentrated in vacuo to afford 20 mg of a pale yellow
colored powdery substance comprising the MI43-37F11 substance. This
powdery substance was dissolved in a small volume of a mixture of
chloroform-methanol (100: 1, V/V), and to the resulting solution was
slowly added n-hexane so that a crystalline substance was deposited. This
crystalline product was collected by filtration and dried to obtain 10 mg
of a crystalline product of the MI43-37F11 substance which exhibited a
melting point of 148.5-149.5.degree. C.
This crystalline product showed a single spot on a silica gel thin layer
chromatography on a silica gel plate (commercially available under a
tradename "Art, 5715", a product of Merck Co., U.S.A.) as developed with
chloroform-methanol (15:1, V/V), revealing that the MI43-37F11 substance
in a pure form was obtained.
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